Proteases

ABSTRACT

Proteases derived from  Nocardiopsis dassonvillei  subsp.  dassonvillei  DSM 43235,  Nocardiopsis prasina  DSM 15649,  Nocardiopsis prasina  (previously  alba ) DSM 14010  Nocardiopsis  sp. DSM 16424,  Nocardiopsis alkaliphila  DSM 44657 and  Nocardiopsis lucentensis  DSM 44048, as well as homologous proteases; their recombinant production in various hosts, including transgenic plants and non-human animals, and their use in animal feed and detergents. The proteases are acid-stable, alkali-stable, and/or thermostable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/490,566filed on Jun. 7, 2012, now pending which is a divisional of U.S.application Ser. No. 11/570,913 filed on Dec. 19, 2006, now U.S. Pat.No. 8,357,408, which is a 35 U.S.C. 371 national application ofPCT/DK2005/000396 filed on Jun. 17, 2005, which claims priority or thebenefit under 35 U.S.C. 119 of Danish application no. PA 2004 00969filed Jun. 21, 2004 and U.S. provisional application No. 60/581,616filed Jun. 21, 2004. The contents of these applications are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to isolated polypeptides having proteaseactivity and isolated nucleic acid sequences encoding the polypeptides.The invention also relates to nucleic acid constructs, vectors, and hostcells, including plant and animal cells, comprising the nucleic acidsequences, as well as methods for producing and using the polypeptides,in particular the use of the polypeptides in animal feed, anddetergents.

BACKGROUND OF THE INVENTION

Proteases derived from Nocardiopsis sp. NRRL 18262 and Nocardiopsisdassonvillei NRRL 18133 are disclosed in WO 88/03947. The DNA and aminoacid sequences of the protease derived from Nocardiopsis sp. NRRL 18262are shown in DK application no. 1996 00013. WO 01/58276 discloses theuse in animal feed of acid-stable proteases related to the proteasederived from Nocardiopsis sp. NRRL 18262, as well as a protease derivedfrom Nocardiopsis alba DSM 14010.

JP 2-255081-A discloses a protease derived from Nocardiopsis sp. strainOPC-210 (FERM P-10508), however without sequence information. The strainis no longer available, as the deposit was withdrawn.

DD 2004328 discloses a proteolytic preparation derived from Nocardiopsisdassonvillei strain ZIMET 43647, however without sequence information.The strain appears to be no longer available.

JP 2003284571-A discloses the amino acid sequence and the correspondingDNA sequence of a protease derived from Nocardiopsis sp. TOA-1 (FERMP-18676). The sequence has been entered in GENESEQP with no. ADF43564.

It is an object of the present invention to provide alternativeproteases, in particular for use in animal feed and/or detergents.

SUMMARY OF THE INVENTION

A number of proteases were cloned, purified and characterized. Theseproteases are designated as follows: Protease L1a derived fromNocardiopsis dassonvillei subsp. dassonvillei DSM 43235 (see SEQ ID NOs.1 and 2); protease L1b derived from Nocardiopsis prasina DSM 15649 (seeSEQ ID NOs: 3 and 4); protease L1c derived from Nocardiopsis prasina(previously alba) DSM 14010 (see SEQ ID NOs: 5 and 6); protease L2aderived from Nocardiopsis sp. DSM 16424 (see SEQ ID NOs: 7 and 8);protease L2b derived from Nocardiopsis alkaliphila DSM 44657 (see SEQ IDNOs: 9 and 10); and protease L2c derived from Nocardiopsis lucentensisDSM 44048 (see SEQ ID NOs: 11 and 12).

In a first aspect, the invention relates to an isolated polypeptidehaving protease activity, selected from the group consisting of: (a) apolypeptide having an amino acid sequence which has a degree of identityto amino acids 1-192 of SEQ ID NO: 6 of at least 71.5%; (b) apolypeptide which is encoded by a nucleic acid sequence which hybridizesunder very high stringency conditions with (i) nucleotides 574-1149 ofSEQ ID NO:5, (ii) a subsequence of (i) of at least 100 nucleotides,and/or (iii) a complementary strand of (i), or (ii); (c) a variant ofthe polypeptide having an amino acid sequence of amino acids 1-192 ofSEQ ID NO: 6 comprising a substitution, deletion, extension, and/orinsertion of one or more amino acids; (d) an allelic variant of (a), or(b); and (e) a fragment of (a), (b), or (d) that has protease activity.

In five alternative aspects, corresponding to the five sets ofparticular embodiments set forth at the end of the present description,the present invention also relates to:

An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-192 of SEQ ID NO: 4 ofat least 69.9%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 574-1149 of SEQ ID NO:3, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-192 of SEQ ID NO: 4 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-192 of SEQ ID NO: 2 ofat least 75.1%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 568-1143 of SEQ ID NO: 1, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-192 of SEQ ID NO: 2 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-189 of SEQ ID NO: 8 ofat least 92.2%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 586-1152 of SEQ ID NO: 7, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-189 of SEQ ID NO: 8 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-189 of SEQ ID NO: 10 ofat least 93.2%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 586-1149 of SEQ ID NO: 9, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-189 of SEQ ID NO: 10 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-189 of SEQ ID NO: 12 ofat least 83.3%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 586-1152 of SEQ ID NO: 11, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-189 of SEQ ID NO: 12 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

The invention also relates to isolated nucleic acid sequences encodingthe above polypeptides and to nucleic acid constructs, vectors, and hostcells comprising the nucleic acid sequences as well as methods forproducing and using the polypeptides, in particular within animal feed,and detergents.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having ProteaseActivity

Polypeptides having protease activity, or proteases, are sometimes alsodesignated peptidases, proteinases, peptide hydrolases, or proteolyticenzymes. Proteases may be of the exo-type that hydrolyses peptidesstarting at either end thereof, or of the endo-type that act internallyin polypeptide chains (endopeptidases). Endopeptidases show activity onN- and C-terminally blocked peptide substrates that are relevant for thespecificity of the protease in question.

The term “protease” is defined herein as an enzyme that hydrolyzespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereof). The EC numberrefers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, SanDiego, Calif., including supplements 1-5 published in Eur. J. Biochem.223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J. Biochem. 237:1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J. Biochem. 264:610-650 (1999); respectively. The nomenclature is regularly supplementedand updated; see, e.g., the World Wide Web (VVVVVV) atchem.qmw.ac.uk/iubmb/enzyme/index.html.

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

In particular embodiments, the proteases of the invention and for useaccording to the invention are selected from the group consisting of:

(a) proteases belonging to the EC 3.4.-.-enzyme group;

(b) Serine proteases belonging to the S group of the above Handbook;

(c) Serine proteases of peptidase family S2A; and/or

(d) Serine proteases of peptidase family 51 E as described in Biochem.J. 290: 205-218 (1993) and in MEROPS protease database, release 6.20,Mar. 24, 2003, (www.merops.ac.uk). The database is described in Rawlingset al., 2002, MEROPS: the protease database. Nucleic Acids Res. 30:343-346.

For determining whether a given protease is a Serine protease, and afamily S2A protease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-values arepH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperaturesare 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95° C.

Examples of protease substrates are casein, such as Azurine-CrosslinkedCasein (AZCL-casein). Three protease assays are described in Examples4-5 herein, either of which can be used to determine protease activity.For the purposes of this invention, the so-called pNA Assay is apreferred assay.

There are no limitations on the origin of the protease of the inventionand/or for use according to the invention. Thus, the term proteaseincludes not only natural or wild-type proteases obtained frommicroorganisms of any genus, but also any mutants, variants, fragmentsetc. thereof exhibiting protease activity, as well as syntheticproteases, such as shuffled proteases, and consensus proteases. Suchgenetically engineered proteases can be prepared as is generally knownin the art, e.g., by Site-directed Mutagenesis, by PCR (using a PCRfragment containing the desired mutation as one of the primers in thePCR reactions), or by Random Mutagenesis. The preparation of consensusproteins is described in, e.g., EP 897985. Gene shuffling is generallydescribed in, e.g., WO 95/22625 and WO 96/00343. Recombination ofprotease genes can be made independently of the specific sequence of theparents by synthetic shuffling as described in Ness et al., 2002, NatureBiotechnology 20(12): 1251-1255. Synthetic oligonucleotides degeneratedin their DNA sequence to provide the possibility of all amino acidsfound in the set of parent proteases are designed and the genesassembled according to the reference. The shuffling can be carried outfor the full length sequence or for only part of the sequence and thenlater combined with the rest of the gene to give a full length sequence.The proteases of SEQ ID NOs: 2, 4, 6, 8, 10, and 12, as well as theNocardiopsis proteases described in the prior documents listed above,are particular examples of such parent proteases which can be subjectedto shuffling as described above, to provide additional proteases of theinvention. The term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by the nucleic acidsequence is produced by the source or by a cell in which the nucleicacid sequence from the source is present. In a preferred embodiment, thepolypeptide is secreted extracellularly.

In a specific embodiment, the protease is a low-allergenic variant,designed to invoke a reduced immunological response when exposed toanimals, including man. The term immunological response is to beunderstood as any reaction by the immune system of an animal exposed tothe protease. One type of immunological response is an allergic responseleading to increased levels of IgE in the exposed animal. Low-allergenicvariants may be prepared using techniques known in the art. For examplethe protease may be conjugated with polymer moieties shielding portionsor epitopes of the protease involved in an immunological response.Conjugation with polymers may involve in vitro chemical coupling ofpolymer to the protease, e.g., as described in WO 96/17929, WO 98/30682,WO 98/35026, and/or WO 99/00489. Conjugation may in addition oralternatively thereto involve in vivo coupling of polymers to theprotease. Such conjugation may be achieved by genetic engineering of thenucleotide sequence encoding the protease, inserting consensus sequencesencoding additional glycosylation sites in the protease and expressingthe protease in a host capable of glycosylating the protease, see, e.g.,WO 00/26354. Another way of providing low-allergenic variants is geneticengineering of the nucleotide sequence encoding the protease so as tocause the proteases to self-oligomerize, effecting that proteasemonomers may shield the epitopes of other protease monomers and therebylowering the antigenicity of the oligomers. Such products and theirpreparation is described, e.g., in WO 96/16177. Epitopes involved in animmunological response may be identified by various methods such as thephage display method described in WO 00/26230 and WO 01/83559, or therandom approach described in EP 561907. Once an epitope has beenidentified, its amino acid sequence may be altered to produce alteredimmunological properties of the protease by known gene manipulationtechniques such as site directed mutagenesis (see, e.g., WO 00/26230, WO00/26354 and/or WO 00/22103) and/or conjugation of a polymer may be donein sufficient proximity to the epitope for the polymer to shield theepitope.

The various aspects of the present invention relate to isolatedpolypeptides having protease activity (for short “proteases”), as wellas the corresponding isolated nucleic acid sequences, said polypeptides,or nucleic acids, respectively, comprising an amino acid sequence, or anucleic acid sequence, respectively, having a certain degree of identityto a specified fragment of an amino acid sequence, or a nucleic acidsequence, respectively, with a specified SEQ ID NO. The fragmentsspecified correspond to the mature polypeptides, or the maturepolypeptide encoding parts of the nucleic acid sequences, respectively.

For purposes of the present invention the degree of identity between twoamino acid sequences, as well as the degree of identity between twonucleotide sequences, is determined by the program “align” which is aNeedleman-Wunsch alignment (i.e., a global alignment). The program isused for alignment of polypeptide, as well as nucleotide sequences. Thedefault scoring matrix BLOSUM50 is used for polypeptide alignments, andthe default identity matrix is used for nucleotide alignments. Thepenalty for the first residue of a gap is −12 for polypeptides and −16for nucleotides. The penalties for further residues of a gap are −2 forpolypeptides, and −4 for nucleotides.

“Align” is part of the FASTA package version v20u6 (see Pearson andLipman, 1988, “Improved Tools for Biological Sequence Analysis”, PNAS85:2444-2448, and Pearson, 1990, “Rapid and Sensitive SequenceComparison with FASTP and FASTA,” Methods in Enzymology 183: 63-98).FASTA protein alignments use the Smith-Waterman algorithm with nolimitation on gap size (see “Smith-Waterman algorithm”, Smith andWaterman, 1981, J. Mol. Biol. 147:195-197).

In particular embodiments, the polypeptide of the invention has a degreeof identity to the mature parts of either of SEQ ID NO: 2, 4, 6, 8, 10,or 12 of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or at least 99%.

In other particular embodiments, the nucleic acid sequence of theinvention has a degree of identity to the mature peptide encoding partof either of SEQ ID NO: 1, 3, 5, 7, 9, or 11 of at least 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or at least 99%.

In still further particular embodiments, the protease of the inventionhas an amino acid sequence that differs by (i) no more than twenty,nineteen, eighteen, seventeen, sixteen, fifteen, fourteen, thirteen,twelve, or no more than eleven amino acids; (ii) no more than ten, nine,eight, seven, six, five, four, three, two, or no more than one aminoacid; (iii) ten, or by nine, or by eight, or by seven, or by six, or byfive amino acids; or (iv) four, or by three, or by two amino acids, orby one amino acid from the mature parts of either of SEQ ID NO: 2, 4, 6,8, 10, and 12.

In a still further particular embodiment, the protease of the inventioncomprises the amino acid sequence of the mature parts of either of SEQID NO: 2, 4, 6, 8, 10, or 12; or is an allelic variant thereof; or afragment thereof that has protease activity.

In a further preferred embodiment, the polypeptides of the presentinvention consist of the mature peptide part of either of SEQ ID NO: 2,4, 6, 8, 10, or 12; or allelic variants thereof; or fragments thereofthat have protease activity.

A fragment of either of SEQ ID NO: 2, 4, 6, 8, 10, or 12 is apolypeptide having one or more amino acids deleted from the amino and/orcarboxyl terminus of these amino acid sequences. In one embodiment afragment contains at least 75 amino acid residues, or at least 100 aminoacid residues, or at least 125 amino acid residues, or at least 150amino acid residues, or at least 160 amino acid residues, or at least165 amino acid residues, or at least 170 amino acid residues, or atleast 175 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The present invention also relates to isolated polypeptides havingprotease activity and which are encoded by nucleic acid sequences whichhybridize under very low, or low, or medium, or medium-high, or high, orvery high stringency conditions with a nucleic acid probe whichhybridizes under the same conditions with (a) either of SEQ ID NO: 1, 3,5, 7, 9, or 11, or the mature peptide encoding parts thereof; (b) asubsequence of (a), or (c) a complementary strand of (a), or (b)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2ndedition, Cold Spring Harbor, N.Y.). In particular embodiments thenucleic acid probe is selected from amongst the nucleic acid sequencesof (a), (b), or (c) above.

The subsequence of (a) may be at least 100 nucleotides, or in anotherembodiment at least 200 nucleotides. Moreover, the subsequence mayencode a polypeptide fragment that has protease activity.

The nucleic acid sequences of either of SEQ ID NO: 1, 3, 5, 7, 9, or 11,or the mature peptide encoding parts thereof, or a subsequence thereof,as well as the amino acid sequences of either of SEQ ID NO: 2, 4, 6, 8,10, or 12, or a fragment thereof, may be used to design a nucleic acidprobe to identify and clone DNA encoding polypeptides having proteaseactivity from strains of the same or different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, preferably at least 25, and more preferably at least 35nucleotides in length. Longer probes can also be used. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA that hybridizes with the probes described aboveand which encodes a polypeptide having protease activity. Genomic orother DNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with either of SEQID NO: 1, 3, 5, 7, 9, or 11, or a subsequence thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleic acid sequencehybridizes to a labeled nucleic acid probe corresponding to the nucleicacid sequence shown in either of these SEQ ID NOs, its complementarystrand, or a subsequence thereof, under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions are detected using X-ray film.

In a particular embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the mature peptide parts of either of SEQ ID NO:2, 4, 6, 8, 10, or 12, or subsequences thereof. In another embodiment,the nucleic acid probe is those nucleotides of either of SEQ ID NO: 1,3, 5, 7, 9, or 11 that correspond to the mature polypeptide codingregions.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency). Preferably, the wash is conducted using either 0.2×SSC,0.1×SSC or 0.02×SSC, the other wash conditions being unamended (i.e.,wash three times, each for 15 minutes; include 0.2% SDS, washingpreferably at least at 45° C. (very low stringency), more preferably atleast at 50° C. (low stringency), more preferably at least at 55° C.(medium stringency), more preferably at least at 60° C. (medium-highstringency), even more preferably at least at 65° C. (high stringency),and most preferably at least at 70° C. (very high stringency)).

For short probes about 15 nucleotides to about 70 nucleotides in length,stringency conditions are defined as prehybridization, hybridization,and washing post-hybridization at 5° C. to 10° C. below the calculatedT_(m) using the calculation according to Bolton and McCarthy (1962,Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 MNaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt'ssolution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate,0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southernblotting procedures.

For short probes about 15 nucleotides to about 70 nucleotides in length,the carrier material is washed once in 6×SSC plus 0.1% SDS for 15minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

The present invention also relates to variants of the polypeptidescomprising the mature parts of either of the amino acid sequences SEQ IDNO: 2, 4, 6, 8, 10, or 12, and comprising a substitution, deletion,and/or insertion of one or more amino acids.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequence of the mature parts of either of SEQ ID NO: 2, 4, 6,8, 10, or 12, by an insertion or deletion of one or more amino acidresidues and/or the substitution of one or more amino acid residues bydifferent amino acid residues. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine).Accordingly, for example, the invention relates to a polypeptide having,or comprising, a sequence as set forth in either of SEQ ID NO: 2, 4, 6,8, 10, or 12, wherein conservative amino acid substitutions comprisereplacements, one for another, among the basic amino acids (arginine,lysine and histidine), among the acidic amino acids (glutamic acid andaspartic acid), among the polar amino acids (glutamine and asparagine),among the hydrophobic amino acids (alanine, leucine, isoleucine andvaline), among the aromatic amino acids (phenylalanine, tryptophan andtyrosine), and among the small amino acids (glycine, alanine, serine,threonine and methionine), or any combination thereof, or activefragments thereof. Amino acid substitutions which do not generally alterthe specific activity are known in the art and are described, forexample, by H. Neurath and R. L. Hill, 1979, In, The Proteins, AcademicPress, New York. The most commonly occurring exchanges are Ala/Ser,Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly,Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, andAsp/Gly as well as these in reverse.

A polypeptide of the present invention may be a bacterial or fungalpolypeptide. The fungal polypeptide may be derived from a filamentousfungus or from a yeast.

In particular embodiments, the polypeptide of the invention is i) abacterial protease; ii) a protease of the phylum Actinobacteria; iii) ofthe class Actinobacteria; iv) of the order Actinomycetales v) of thefamily Nocardiopsaceae; vi) of the genus Nocardiopsis; and/or a proteasederived from vii) Nocardiopsis species such as Nocardiopsis alba,Nocardiopsis alkaliphila, Nocardiopsis antarctica, Nocardiopsis prasina,Nocardiopsis composta, Nocardiopsis exhalans, Nocardiopsis halophila,Nocardiopsis halotolerans, Nocardiopsis kunsanensis, Nocardiopsislisteri, Nocardiopsis lucentensis, Nocardiopsis metallicus, Nocardiopsissynnemataformans, Nocardiopsis trehalosi, Nocardiopsis tropica,Nocardiopsis umidischolae, Nocardiopsis xinjiangensis, or Nocardiopsisdassonvillei, for example from either of Nocardiopsis dassonvilleisubsp. dassonvillei DSM 43235, Nocardiopsis prasina DSM 15649,Nocardiopsis prasina (previously alba) DSM 14010, Nocardiopsis sp. DSM16424, Nocardiopsis alkaliphila DSM 44657, or from Nocardiopsislucentensis DSM 44048.

In a particular embodiment, the protease derives from Nocardiopsis alba,Nocardiopsis alkaliphila, Nocardiopsis dassonvillei, Nocardiopsislucentensis, Nocardiopsis prasina, or Nocardiopsis sp.

The above taxonomy is according to the chapter: The road map to theManual by G. M. Garrity & J. G. Holt in Bergey's Manual of SystematicBacteriology, 2001, second edition, volume 1, David R. Bone, Richard W.Castenholz.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism. Once anucleic acid sequence encoding a polypeptide has been detected with theprobe(s), the sequence may be isolated or cloned by utilizing techniqueswhich are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

As defined herein, an “isolated” polypeptide is a polypeptide which isessentially free of other non-protease polypeptides, e.g., at leastabout 20% pure, preferably at least about 40% pure, more preferablyabout 60% pure, even more preferably about 80% pure, most preferablyabout 90% pure, and even most preferably about 95% pure, as determinedby SDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleic acid sequence (or a portion thereof) encodinganother polypeptide to a nucleic acid sequence (or a portion thereof) ofthe present invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

In a particular embodiment, the polypeptides of the invention areacid-stable. For the present purposes, the term acid-stable means thatthe residual activity after 2 hours of incubation at pH 2.0, pH 2.5 orpH 3.0 and 37° C., is at least 50%, as compared to the residual activityof a corresponding sample incubated for 2 hours at pH 9.0 and 5° C. In aparticular embodiment, the residual activity is at least 55%, 60%, 65%,70%, 75%, 80%, 85%, or at least 90%. A suitable assay for determiningacid-stability is the pH-stability assay of Example 2.

In another particular embodiment, the polypeptides of the invention arealkali-stable. For the present purposes, the term alkali-stable meansthat the residual activity after 2 hours of incubation at pH 12.0 and37° C., is at least 85%, as compared to the residual activity of acorresponding sample incubated for 2 hours at pH 9.0 and 5° C. In aparticular embodiment, the residual activity is at least 86%, 87%, 88%,89%, 90%, 91%, or at least 92%. A suitable assay for determiningalkali-stability is the pH-stability assay of Example 4.

In still further particular embodiments, the polypeptides of theinvention and for use according to the invention have i) a relativeactivity at 15° C. and pH 9 of at least 0.02, 0.04, 0.06, 0.08, 0.10, orat least 0.11; ii) a relative activity at 25° C. and pH 9 of at least0.05, 0.10, 0.15, or at least 0.17; and/or iii) a relative activity at37° C. and pH 9 of at least 0.05, 0.10, 0.15, 0.20, 0.25, or at least0.30. The temperature-profile test of Example 4 is used for thesedeterminations.

In still further particular embodiments, the polypeptides of theinvention have a Tm, as determined by DSC, of at least 76.6° C., or ofat least 77, 78, or at least 78.2° C. Tm is determined at pH 7.0 asdescribed in Example 7.

In an additional particular embodiment the protease of the inventionexhibits a specific activity on haemoglobin at pH 7.5 and 25° C. of atleast 38.4 AU/g. The specific activity may be determined as described inExample 5. The protease of the invention may exhibit a specific activityof at least 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 49.8, or atleast 50 AU/g.

In a further particular embodiment, the protease of the invention iscapable of improving, by at least 13% as compared to the blank, thelevel of digestible protein of a maize/soybean meal diet (SBM:Maize=2:3(w/w)) in a monogastric in vitro digestion model. The model includes agastric digestion step (1.0 hour at pH 3.0 and 40° C.), and a subsequentintestinal digestion step (4.5 hours at pH 6.8 and 40° C.). The modelalso includes addition of pepsin (3000 U/g, in the gastric digestionstep), and of pancreatin (8 mg/g, in the intestinal digestion step). Theprotease dosage is 100 mg protease enzyme protein (EP) per kg of diet. Asuitable model is described in Example 8. The level of improvement maybe at least 14%, 15%, or at least 16%.

In still further particular embodiments, the invention excludes theprotease derived from (i) Nocardiopsis dassonvillei NRRL 18133 which isdisclosed in WO 88/03947; (ii) Nocardiopsis sp. strain OPC-210 (FERMP-10508) which is disclosed in JP 2-255081-A; and/or (iii) the proteasederived from strain ZIMET 43647 of the species Nocardiopsis dassonvilleiwhich is disclosed in DD 2004328.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequencesthat encode a polypeptide of the present invention. Particular nucleicacid sequences of the invention are (i) nucleotides 1-1143, 1-87,88-567, and 568-1143 of SEQ ID NO: 1; (ii) nucleotides 1-1149, 1-87,88-573, and 574-1149 of SEQ ID NO: 3; (iii) nucleotides 1-1149, 1-87,88-573, and 574-1149 of SEQ ID NO: 5; (iv) nucleotides 1-1152, 1-87,88-585, and 586-1152 of SEQ ID NO: 7; (v) nucleotides 1-1149, 1-87,88-585, and 586-1149 of SEQ ID NO: 9; and (vi) nucleotides 1-1152, 1-87,88-585, and 586-1152 of SEQ ID NO: 11; and (vii) any combinationthereof.

Particularly preferred nucleotides are (i) nucleotides 568-1143 of SEQID NO: 1; (ii) nucleotides 574-1149 of SEQ ID NO: 3; (iii) nucleotides574-1149 of SEQ ID NO: 5; (iv) nucleotides 586-1152 of SEQ ID NO: 7; (v)nucleotides 586-1149 of SEQ ID NO: 9; and (vi) nucleotides 586-1152 ofSEQ ID NO: 11; corresponding to the mature polypeptide encoding parts orregions.

The present invention also encompasses nucleic acid sequences whichencode a polypeptide having the mature parts of either of SEQ ID NO: 2,4, 6, 8, 10 or 12, which differ from the corresponding parts of SEQ IDNO: 1, 3, 5, 7, 9, or 11, respectively, by virtue of the degeneracy ofthe genetic code. The present invention also relates to subsequences ofeither of SEQ ID NO: 1, 3, 5, 7, 9, or 11 which encode fragments of SEQID NO: 2, 4, 6, 8, 10 or 12, respectively, and which have proteaseactivity.

A subsequence of either of SEQ ID NO: 1, 3, 5, 7, 9, or 11 is a nucleicacid sequence encompassed by SEQ ID NO: 1, 3, 5, 7, 9 or 11, except thatone or more nucleotides from the 5′ and/or 3′ end have been deleted.Preferably, a subsequence contains at least 100, 125, 150, 175, 200, orat least 225 nucleotides, more preferably at least 300 nucleotides, evenmore preferably at least 325, 350, 375, 400, 425, 450, 475, 500, 525,550, or at least 560 nucleotides.

The present invention also relates to nucleotide sequences which have adegree of identity to the mature peptide encoding parts of either of SEQID NO: 1, 3, 5, 7, 9, or 11 of at least 77.7%, preferably of at least78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

For determining the degree of nucleotide identity, the program “align”is used which is referred to above.

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in any of the nucleotides of (i)-(vi)listed above, preferably the mature peptide encoding parts thereof, inwhich the mutant nucleic acid sequence encodes a polypeptide which (i)consists of the amino acid sequences of either of SEQ ID NO: 2, 4, 6, 8,10, or 12, preferably the mature peptide parts thereof, or (ii) is avariant of any of the sequences of (i), wherein the variant comprises asubstitution, deletion, and/or insertion of one or more amino acids, or(iii) is an allelic variant of any of the sequences of (i), or (iv) is afragment of any of the sequences of (i).

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Nocardiopsis, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum,allergenicity, or the like. The variant sequence may be constructed onthe basis of the nucleic acid sequence presented as the polypeptideencoding part, or mature peptide encoding part, of either of SEQ ID NO:1, 3, 5, 7, 9 or 11, e.g., a subsequence thereof, and/or by introductionof nucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the protease, or by introduction of nucleotidesubstitutions which may give rise to a different amino acid sequence.For a general description of nucleotide substitution, see, e.g., Ford etal., 1991, Protein Expression and Purification 2: 95-107. Low-allergenicpolypeptides can, e.g., be prepared as described above.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor protease activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-protease interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated nucleic acid sequencesencoding a polypeptide of the present invention, which hybridize undervery low stringency conditions, preferably low stringency conditions,more preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with anucleic acid probe which hybridizes under the same conditions with thenucleic acid sequence of either of SEQ ID NO: 1, 3, 5, 7, 9, or 11,preferably the mature peptide encoding parts thereof, or a complementarystrand; or an allelic variant; or a subsequence thereof (Sambrook etal., 1989, supra), as defined herein.

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with any of thenucleotides mentioned under (i)-(vi) above, preferably the maturepeptide encoding parts thereof, or a subsequence, or a complementarystrand thereof; and (b) isolating the nucleic acid sequence.

Methods for Producing Mutant Nucleic Acid Sequences

The present invention further relates to methods for producing a mutantnucleic acid sequence, comprising introducing at least one mutation intothe mature polypeptide coding parts of either of SEQ ID NO: 1, 3, 5, 7,9, or 11, or a subsequence thereof, wherein the mutant nucleic acidsequence encodes a polypeptide which consists of the mature peptide ofSEQ ID NO: 2, 4, 6, 8, 10, or 12, respectively; or a fragment thereofwhich has protease activity.

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure that utilizes a supercoiled, doublestranded DNA vector with an insert of interest and two synthetic primerscontaining the desired mutation. The oligonucleotide primers, eachcomplementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with Dpnl which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention. The term “coding sequence” is defined herein as anucleic acid sequence that directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) located just upstream of the open reading frame atthe 5′ end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′ end of the mRNA. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

The term “control sequences” is defined herein to include all componentsthat are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence that is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences that mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican 242: 74-94 (1980); and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

Preferred terminators for bacterial host cells, such as a Bacillus hostcell, are the terminators from Bacillus licheniformis alpha-amylase gene(amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM),or the Bacillus amyloliquefaciens alpha-amylase gene (amyQ).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA.

Any polyadenylation sequence which is functional in the host cell ofchoice may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformis alpha-amylase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are described by Simonenand Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

In a preferred embodiment, the signal peptide coding region is thesignal peptide coding region of either of SEQ ID NO: 1, 3, 5, 7, 9, or11.

In another preferred embodiment, the propeptide coding region is thepropeptide coding regions of either of SEQ ID NO: 1, 3, 5, 7, 9, or 11.

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in a filamentous fungal host cell include, but are notlimited to, amdS (acetamidase), argB (ornithine carbamoyltransferase),bar (phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or non homologous recombination. Alternatively, the vectormay contain additional nucleic acid sequences for directing integrationby homologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

The protease may also be co-expressed together with at least one otherenzyme of interest for animal feed, such as an amylase; phytase;xylanase; galactanase; alpha-galactosidase; protease, phospholipase;and/or a beta-glucanase.

The enzymes may be co-expressed from different vectors, from one vector,or using a mixture of both techniques. When using different vectors, thevectors may have different selectable markers, and different origins ofreplication. When using only one vector, the genes can be expressed fromone or more promoters. If cloned under the regulation of one promoter(di- or multi-cistronic), the order in which the genes are cloned mayaffect the expression levels of the proteins. The protease may also beexpressed as a fusion protein, i.e., that the gene encoding protease hasbeen fused in frame to the gene encoding another protein. This proteinmay be another enzyme or a functional domain from another enzyme.

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, or aStreptomyces cell, or cells of lactic acid bacteria; or gram negativebacteria such as E. coli and Pseudomonas sp. Lactic acid bacteriainclude, but are not limited to, species of the genera Lactococcus,Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, andEnterococcus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a non-human animal cell, aninsect cell, a plant cell, or a fungal cell.

In one particular embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In another particular embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

Examples of filamentous fungal host cells are cells of species of, butnot limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, orTrichoderma.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp.182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide. In a preferred embodiment, the strain isof the phylum Actinobacteria, preferably of the class Actinobacteria,more preferably of the order Actinomycetales, even more preferably ofthe family Nocardiopsaceae, and most preferably of the genusNocardiopsis, for example any of the Nocardiopsis species, such as thespecific strains listed hereinbefore.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequencecomprising at least one mutation in the mature peptide encoding parts ofeither of SEQ ID NO: 1, 3, 5, 7, 9, or 11, in which the mutant nucleicacid sequence encodes a polypeptide which (i) consists of the maturepeptides of either of SEQ ID NO: 2, 4, 6, 8, 10, or 12, respectively; or(ii) is a variant of any of the sequences of (i), wherein the variantcomprises a substitution, deletion, and/or insertion of one or moreamino acids, or (iii) is an allelic variant of any of the sequences of(i), or (iv) is a fragment of any of the sequences of (i).

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of a protease product, ordisappearance of a protease substrate. For example, a protease assay maybe used to determine the activity of the polypeptide as describedherein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleic acid sequenceencoding a polypeptide having protease activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

In a particular embodiment, the polypeptide is targeted to the endospermstorage vacuoles in seeds. This can be obtained by synthesizing it as aprecursor with a suitable signal peptide, see Horvath et al. in PNAS97(4): 1914-1919 (Feb. 15, 2000).

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot) or engineered variants thereof. Examples of monocot plantsare grasses, such as meadow grass (blue grass, Poa), forage grass suchas Festuca, Lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilizedhybrid of wheat (Triticum) and rye (Secale), and maize (corn). Examplesof dicot plants are tobacco, legumes, such as sunflower (Helianthus),cotton (Gossypium), lupins, potato, sugar beet, pea, bean and soybean,and cruciferous plants (family Brassicaceae), such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.Low-phytate plants as described, e.g., in U.S. Pat. No. 5,689,054 andU.S. Pat. No. 6,111,168 are examples of engineered plants.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers, as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyma, vascular tissues, meristems.Also specific plant cell compartments, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part. Likewise, plant parts such as specifictissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific cell compartment, tissue or plant partsuch as seeds or leaves. Regulatory sequences are, for example,described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the following promoters may be used: The35S-CaMV promoter (Franck et al., 1980, Cell 21: 285-294), the maizeubiquitin 1 (Christensen A H, Sharrock R A and Quail 1992. Maizepolyubiquitin genes: structure, thermal perturbation of expression andtranscript splicing, and promoter activity following transfer toprotoplasts by electroporation), or the rice actin 1 promoter (PlantMol. Biol. 18, 675-689.; Zhang et al., 1991, Analysis of rice Act1 5′region activity in transgenic rice plants. Plant Cell 3, 1155-1165).Organ-specific promoters may be, for example, a promoter from storagesink tissues such as seeds, potato tubers, and fruits (Edwards &Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sinktissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24:863-878), a seed specific promoter such as the glutelin, prolamin,globulin, or albumin promoter from rice (Wu et al., 1998, Plant and CellPhysiology 39: 885-889), a Vicia faba promoter from the legumin B4 andthe unknown seed protein gene from Vicia faba (Conrad et al., 1998,Journal of Plant Physiology 152: 708-711), a promoter from a seed oilbody protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, PlantPhysiology 102: 991-1000, the chlorella virus adenine methyltransferasegene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), or the aldP gene promoter from rice (Kagaya et al., 1995,Molecular and General Genetics 248: 668-674), or a wound induciblepromoter such as the potato pin2 promoter (Xu et al., 1993, PlantMolecular Biology 22: 573-588). Likewise, the promoter may be inducibleby abiotic treatments such as temperature, drought or alterations insalinity or inducible by exogenously applied substances that activatethe promoter, e.g., ethanol, oestrogens, plant hormones like ethylene,abscisic acid, gibberellic acid, and/or heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of the protease in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

Still further, the codon usage may be optimized for the plant species inquestion to improve expression (see Horvath et al. referred to above).

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38), andit can also be used for transforming monocots, although othertransformation methods are more often used for these plants. Presently,the method of choice for generating transgenic monocots, supplementingthe Agrobacterium approach, is particle bombardment (microscopic gold ortungsten particles coated with the transforming DNA) of embryonic callior developing embryos (Christou, 1992, Plant Journal 2: 275-281;Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al.,1992, Bio/Technology 10: 667-674). An alternative method fortransformation of monocots is based on protoplast transformation asdescribed by Omirulleh et al., 1993, Plant Molecular Biology 21:415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using,e.g., co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having protease activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Animals

The present invention also relates to a transgenic, non-human animal andproducts or elements thereof, examples of which are body fluids such asmilk and blood, organs, flesh, and animal cells. Techniques forexpressing proteins, e.g., in mammalian cells, are known in the art,see, e.g., the handbook Protein Expression: A Practical Approach,Higgins and Hames (eds), Oxford University Press (1999), and the threeother handbooks in this series relating to Gene Transcription, RNAprocessing, and Post-translational Processing. Generally speaking, toprepare a transgenic animal, selected cells of a selected animal aretransformed with a nucleic acid sequence encoding a polypeptide havingprotease activity of the present invention so as to express and producethe polypeptide. The polypeptide may be recovered from the animal, e.g.,from the milk of female animals, or the polypeptide may be expressed tothe benefit of the animal itself, e.g., to assist the animal'sdigestion. Examples of animals are mentioned below in the section headedAnimal Feed.

To produce a transgenic animal with a view to recovering protease fromthe milk of the animal, a gene encoding the protease may be insertedinto the fertilized eggs of an animal in question, e.g., by use of atransgene expression vector which comprises a suitable milk proteinpromoter, and the gene encoding protease. The transgene expressionvector is microinjected into fertilized eggs, and preferably permanentlyintegrated into the chromosome. Once the egg begins to grow and divide,the potential embryo is implanted into a surrogate mother, and animalscarrying the transgene are identified. The resulting animal can then bemultiplied by conventional breeding. The polypeptide may be purifiedfrom the animal's milk, see, e.g., Meade, H. M. et al (1999): Expressionof recombinant proteins in the milk of transgenic animals, Geneexpression systems: Using nature for the art of expression. J. M.Fernandez and J. P. Hoeffler (eds.), Academic Press.

In the alternative, in order to produce a transgenic non-human animalthat carries in the genome of its somatic and/or germ cells a nucleicacid sequence including a heterologous transgene construct including atransgene encoding protease, the transgene may be operably linked to afirst regulatory sequence for salivary gland specific expression ofprotease, as disclosed in WO 00/64247.

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptides orpolypeptide compositions of the invention.

Animal Feed

The present invention is also directed to methods for using thepolypeptides having protease activity in animal feed, as well as to feedcompositions and feed additives comprising the polypeptides of theinvention.

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goats, horses, and cattle, e.g.,beef cattle, cows, and young calves. In a particular embodiment, theanimal is a non-ruminant animal. Non-ruminant animals includemono-gastric animals, e.g., pigs or swine (including, but not limitedto, piglets, growing pigs, and sows); poultry such as turkeys, ducks andchicken (including but not limited to broiler chicks, layers); youngcalves; and fish (including but not limited to salmon, trout, tilapia,catfish and carps; and crustaceans (including but not limited to shrimpsand prawns).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the protease can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the protease, in the form in which it isadded to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the protease preparation is atleast 50% pure as determined by Size-exclusion chromatography (seeExample 12 of WO 01/58275). In other particular embodiments the proteasepreparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95%pure as determined by this method.

A well-defined protease preparation is advantageous. For instance, it ismuch easier to dose correctly to the feed a protease that is essentiallyfree from interfering or contaminating other proteases. The term dosecorrectly refers in particular to the objective of obtaining consistentand constant results, and the capability of optimising dosage based uponthe desired effect.

For the use in animal feed, however, the protease need not be that pure;it may, e.g., include other enzymes, in which case it could be termed aprotease preparation.

The protease preparation can be (a) added directly to the feed (or useddirectly in a protein treatment process), or (b) it can be used in theproduction of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original protease preparation, whether used according to(a) or (b) above.

Protease preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the protease is produced by traditionalfermentation methods.

Such protease preparation may of course be mixed with other enzymes.

In a further particular embodiment, the protease for use according tothe invention is capable of solubilizing proteins according to the invitro model of Example 8 herein.

The protein may be an animal protein, such as meat and bone meal, and/orfish meal; or it may be a vegetable protein.

The term vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.,beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

The treatment according to the invention of proteins with at least oneprotease of the invention results in an increased solubilization ofproteins, as compared to the blank. At least 101%, or 102%, 103%, 104%,105%, 106%, or at least 107% solubilized protein may be obtainable usingthe proteases of the invention, reference being had to the in vitromodel of Example 8 herein. The term solubilization of proteins basicallymeans bringing protein(s) into solution. Such solubilization may be dueto protease-mediated release of protein from other components of theusually complex natural compositions such as feed. Solubilization can bemeasured as an increase in the amount of soluble proteins, by referenceto a sample with no protease treatment (see Example 8).

The treatment according to the invention of proteins with at least oneprotease of the invention results in an increased digestibility ofproteins, as compared to the blank. At least 101%, or 102%, 103%, 104%,105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, or atleast 116% digestible protein may be obtainable using the proteases ofthe invention, reference being had to the in vitro model of Example 8herein.

In a particular embodiment of a treatment process the protease(s) inquestion is affecting (or acting on, or exerting its solubilizinginfluence on) the proteins, such as vegetable proteins or proteinsources. To achieve this, the protein or protein source is typicallysuspended in a solvent, e.g., an aqueous solvent such as water, and thepH and temperature values are adjusted paying due regard to thecharacteristics of the enzyme in question. For example, the treatmentmay take place at a pH-value at which the activity of the actualprotease is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or atleast 90%. Likewise, for example, the treatment may take place at atemperature at which the activity of the actual protease is at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%. The abovepercentage activity indications are relative to the maximum activities.The enzymatic reaction is continued until the desired result isachieved, following which it may or may not be stopped by inactivatingthe enzyme, e.g., by a heat-treatment step.

In another particular embodiment of a treatment process of theinvention, the protease action is sustained, meaning, e.g., that theprotease is added to the proteins, but its solubilizing influence is soto speak not switched on until later when desired, once suitablesolubilizing conditions are established, or once any enzyme inhibitorsare inactivated, or whatever other means could have been applied topostpone the action of the enzyme.

In one embodiment the treatment is a pre-treatment of animal feed orproteins for use in animal feed, i.e., the proteins are solubilizedbefore intake.

The term improving the nutritional value of an animal feed meansimproving the availability of the proteins, thereby leading to increasedprotein extraction, higher protein yields, and/or improved proteinutilisation. The nutritional value of the feed is therefore increased,and the growth rate and/or weight gain and/or feed conversion (i.e., theweight of ingested feed relative to weight gain) of the animal is/areimproved.

The protease can be added to the feed in any form, be it as a relativelypure protease, or in admixture with other components intended foraddition to animal feed, i.e., in the form of animal feed additives,such as the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives,e.g., premixes.

Apart from the protease of the invention, the animal feed additives ofthe invention contain at least one fat-soluble vitamin, and/or at leastone water soluble vitamin, and/or at least one trace mineral, and/or atleast one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.,carotenoids such as beta-carotene, astaxanthin, and lutein; aromacompounds; stabilisers; antimicrobial peptides; polyunsaturated fattyacids; reactive oxygen generating species; and/or at least one otherenzyme selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26); xylanase(EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC3.2.1.22); protease (EC 3.4.-.-), phospholipase A1 (EC 3.1.1.32);phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5);phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase suchas, for example, alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC3.2.1.4 or EC 3.2.1.6).

In a particular embodiment these other enzymes are well-defined (asdefined above for protease preparations).

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a protease of the invention, is ananimal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed).

This is so in particular for premixes.

The following are non-exclusive lists of examples of these components:Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g., vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g., Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

In a still further embodiment, the animal feed additive of the inventioncomprises at least one of the below vitamins, preferably to provide anin-feed-concentration within the ranges specified in the below table I(for piglet diets, and broiler diets, respectively).

TABLE I Typical vitamin recommendations Vitamin Piglet diet Broiler dietVitamin A 10,000-15,000 IU/kg feed 8-12,500 IU/kg feed Vitamin D31800-2000 IU/kg feed 3000-5000 IU/kg feed Vitamin E 60-100 mg/kg feed150-240 mg/kg feed Vitamin K3 2-4 mg/kg feed 2-4 mg/kg feed Vitamin B12-4 mg/kg feed 2-3 mg/kg feed Vitamin B2 6-10 mg/kg feed 7-9 mg/kg feedVitamin B6 4-8 mg/kg feed 3-6 mg/kg feed Vitamin B12 0.03-0.05 mg/kgfeed 0.015-0.04 mg/kg feed Niacin 30-50 mg/kg feed 50-80 mg/kg feed(Vitamin B3) Pantothenic 20-40 mg/kg feed 10-18 mg/kg feed acid Folicacid 1-2 mg/kg feed 1-2 mg/kg feed Biotin 0.15-0.4 mg/kg feed 0.15-0.3mg/kg feed Choline 200-400 mg/kg feed 300-600 mg/kg feed chloride

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterized as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterized as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg.

WO 01/58275 corresponds to U.S. Ser. No. 09/779,334 which is herebyincorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneprotease as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e., Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein as defined above. It may alsocontain animal protein, such as Meat and Bone Meal, and/or Fish Meal,typically in an amount of 0-25%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can, e.g., be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, a solidenzyme formulation is typically added before or during the mixing step;and a liquid enzyme preparation is typically added after the pelletingstep. The enzyme may also be incorporated in a feed additive or premix.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, for example in the range of0.5-25 mg enzyme protein per kg animal diet.

The protease should of course be applied in an effective amount, i.e.,in an amount adequate for improving solubilization, digestibility,and/or improving nutritional value of feed. It is at presentcontemplated that the enzyme is administered in one or more of thefollowing amounts (dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50;5-100; 10-100; 0.05-50; or 0.10-10—all these ranges being in mg proteaseprotein per kg feed (ppm).

For determining mg protease protein per kg feed, the protease ispurified from the feed composition, and the specific activity of thepurified protease is determined using a relevant assay (see underprotease activity, substrates, and assays). The protease activity of thefeed composition as such is also determined using the same assay, and onthe basis of these two determinations, the dosage in mg protease proteinper kg feed is calculated.

The same principles apply for determining mg protease protein in feedadditives. Of course, if a sample is available of the protease used forpreparing the feed additive or the feed, the specific activity isdetermined from this sample (no need to purify the protease from thefeed composition or the additive).

Detergent Compositions

The protease of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the protease of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas another protease, such as alkaline proteases from Bacillus, a lipase,a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., alaccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258068 and EP 305216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218272), P.cepacia (EP 331376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochimica et Biophysica Acta 1131: 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Otherexamples are lipase variants such as those described in WO 92/05249, WO94/01541, EP 407225, EP 260105, WO 95/35381, WO 96/00292, WO 95/30744,WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.Preferred commercially available lipase enzymes include Lipolase™ andLipolase Ultra™ (Novozymes A/S). Suitable amylases (alpha- and/or beta-)include those of bacterial or fungal origin. Chemically modified orprotein engineered mutants are included. Amylases include, for example,alpha-amylases obtained from Bacillus, e.g., a special strain of B.licheniformis, described in more detail in GB 1,296,839. Examples ofuseful amylases are the variants described in WO 94/02597, WO 94/18314,WO 95/26397, WO 96/23873, WO 97/43424, WO 00/60060, and WO 01/66712,especially the variants with substitutions in one or more of thefollowing positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181,188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.Commercially available amylases are Natalase™, Supramyl™, Stainzyme™,Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ andPurastar™ (from Genencor International Inc.).

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0495257, EP 531372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0531315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and WO99/01544. Commercially available cellulases include Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257. Commerciallyavailable peroxidases include Guardzyme™ (Novozymes).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, e.g., as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, e.g., WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01-100 mg of enzyme protein per liter of washliquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor,in particular 0.1-1 mg of enzyme protein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202.

Particular Embodiments

The invention also relates to the following particular embodiments, inwhat follows numbered 1-21 and 42-121:

1. An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-192 of SEQ ID NO: 4 ofat least 69.9%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 574-1149 of SEQ ID NO:3, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-192 of SEQ ID NO: 4 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

2. The polypeptide of embodiment 1 which comprises any one of thefollowing proteases: (a) amino acids 1-192 of SEQ ID NO: 6; (b) aminoacids 1-189 of SEQ ID NO: 10; (c) amino acids 1-192 of SEQ ID NO: 2; or(d) amino acids 1-192 of SEQ ID NO: 4.

3. An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes a polypeptide having protease activity, and which (a)encodes the polypeptide of any one of embodiments 1-2; (b) hybridizesunder low stringency conditions with (i) nucleotides 574-1149 of SEQ IDNO:3, (ii) a subsequence of (i) of at least 100 nucleotides, and/or(iii) a complementary strand of (i), or (ii); and/or (c) has a degree ofidentity to nucleotides 574-1149 of SEQ ID NO: 3 of at least 77.7%.

4. The nucleic acid sequence of embodiment 3 which comprises any one ofthe following protease-encoding nucleic acid sequences: (a) nucleotides574-1149 of SEQ ID NO: 3; (b) nucleotides 574-1149 of SEQ ID NO: 5; (c)nucleotides 586-1152 of SEQ ID NO: 7; or (d) nucleotides 568-1143 of SEQID NO: 1.

5. An isolated nucleic acid sequence produced by (a) hybridizing a DNAunder low stringency conditions with (i) nucleotides 574-1149 of SEQ IDNO:3; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) acomplementary strand of (i), or (ii); and (b) isolating the nucleic acidsequence.

6. A nucleic acid construct comprising the nucleic acid sequence of anyone of embodiments 3-5 operably linked to one or more control sequencesthat direct the production of the polypeptide in a suitable expressionhost.

7. A recombinant expression vector comprising the nucleic acid constructof embodiment 6.

8. A recombinant host cell comprising the nucleic acid construct ofembodiment 6 or the vector of embodiment 7.

9. A transgenic plant, or plant part, capable of expressing thepolypeptide of any one of embodiments 1-2.

10. A transgenic, non-human animal, or products or elements thereof,being capable of expressing the polypeptide of any one of embodiments1-2.

11. A method for producing a polypeptide of any one of embodiments 1-2,the method comprising (a) cultivating a recombinant host cell ofembodiment 8 to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide.

12. A method for producing a polypeptide of any one of embodiments 1-2,the method comprising (a) cultivating any one of the following strains:(i) Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235, (ii)Nocardiopsis prasina DSM 15649, (iii) Nocardiopsis prasina DSM 14010, or(iv) Nocardiopsis alkaliphila DSM 44657; and (b) recovering thepolypeptide.

13. Use of at least one protease of any one of embodiments 1-2 (i) inanimal feed; (ii) in animal feed additives; (iii) in the preparation ofa composition for use in animal feed; (iv) for improving the nutritionalvalue of an animal feed; (v) for increasing digestible and/or solubleprotein in animal feed; (vi) for increasing the degree of hydrolysis ofproteins in animal diets; and/or (vii) for the treatment of proteins.

14. A method for improving the nutritional value of an animal feed,wherein at least one protease of any one of embodiments 1-2 is added tothe feed.

15. An animal feed additive comprising (a) at least one protease of anyone of embodiments 1-2; and (b) at least one fat-soluble vitamin, and/or(c) at least one water-soluble vitamin, and/or (d) at least one tracemineral.

16. The animal feed additive of embodiment 15, which further comprisesamylase; phytase; xylanase; galactanase; alpha-galactosidase; protease,phospholipase; and/or beta-glucanase.

17. An animal feed having a crude protein content of 50 to 800 g/kg andcomprising at least one protease of any one of embodiments 1-2.

18. A method for the treatment of proteins, comprising the step ofadding at least one protease of any one of embodiments 1-2 to at leastone protein or protein source.

19. The method of embodiment 18, wherein soybean is included amongst theat least one protein source.

20. Use of at least one protease of any one of embodiments 1-2 indetergents.

21. Nocardiopsis sp. DSM 16424.

42. An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-192 of SEQ ID NO: 2 ofat least 75.1%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 568-1143 of SEQ ID NO: 1, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-192 of SEQ ID NO: 2 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

43. The polypeptide of embodiment 42 which comprises any one of thefollowing proteases: (a) amino acids 1-192 of SEQ ID NO: 6; (b) aminoacids 1-192 of SEQ ID NO: 2; or (c) amino acids 1-192 of SEQ ID NO: 4.

44. An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes a polypeptide having protease activity, and which (a)encodes the polypeptide of any one of embodiments 42-43; (b) hybridizesunder low stringency conditions with (i) nucleotides 568-1143 of SEQ IDNO: 1, (ii) a subsequence of (i) of at least 100 nucleotides, and/or(iii) a complementary strand of (i), or (ii); and/or (c) has a degree ofidentity to nucleotides 568-1143 of SEQ ID NO: 1 of at least 81.2%.

45. The nucleic acid sequence of embodiment 3 or 44 which comprises anyone of the following protease-encoding nucleic acid sequences: (a)nucleotides 574-1149 of SEQ ID NO: 3; (b) nucleotides 574-1149 of SEQ IDNO: 5; or (c) nucleotides 568-1143 of SEQ ID NO: 1.

46. An isolated nucleic acid sequence produced by (a) hybridizing a DNAunder low stringency conditions with (i) nucleotides 568-1143 of SEQ IDNO: 1; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) acomplementary strand of (i), or (ii); and (b) isolating the nucleic acidsequence.

47. A nucleic acid construct comprising the nucleic acid sequence of anyone of embodiments 44-46 operably linked to one or more controlsequences that direct the production of the polypeptide in a suitableexpression host.

48. A recombinant expression vector comprising the nucleic acidconstruct of embodiment 47.

49. A recombinant host cell comprising the nucleic acid construct ofembodiment 47 or the vector of embodiment 48.

50. A transgenic plant, or plant part, capable of expressing thepolypeptide of any one of embodiments 42-43.

51. A transgenic, non-human animal, or products or elements thereof,being capable of expressing the polypeptide of any one of embodiments42-43.

52. A method for producing a polypeptide of any one of embodiments42-43, the method comprising (a) cultivating a recombinant host cell ofembodiment 49 to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide.

53. A method for producing a polypeptide of any one of embodiments42-43, the method comprising (a) cultivating any one of the followingstrains: (i) Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235,(ii) Nocardiopsis prasina DSM 15649, or (iii) Nocardiopsis prasina DSM14010; and (b) recovering the polypeptide.

54. Use of at least one protease of any one of embodiments 42-43 (i) inanimal feed; (ii) in animal feed additives; (iii) in the preparation ofa composition for use in animal feed; (iv) for improving the nutritionalvalue of an animal feed; (v) for increasing digestible and/or solubleprotein in animal feed; (vi) for increasing the degree of hydrolysis ofproteins in animal diets; and/or (vii) for the treatment of proteins.

55. A method for improving the nutritional value of an animal feed,wherein at least one protease of any one of embodiments 42-43 is addedto the feed.

56. An animal feed additive comprising (a) at least one protease of anyone of embodiments 42-43; and (b) at least one fat-soluble vitamin,and/or (c) at least one water-soluble vitamin, and/or (d) at least onetrace mineral.

57. The animal feed additive of embodiment 56, which further comprisesamylase; phytase; xylanase; galactanase; alpha-galactosidase; protease,phospholipase; and/or beta-glucanase.

58. An animal feed having a crude protein content of 50 to 800 g/kg andcomprising at least one protease of any one of embodiments 42-43.

59. A method for the treatment of proteins, comprising the step ofadding at least one protease of any one of embodiments 42-43 to at leastone protein or protein source.

60. The method of embodiment 59, wherein soybean is included amongst theat least one protein source.

61. Use of at least one protease of any one of embodiments 42-43 indetergents.

62. An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-189 of SEQ ID NO: 8 ofat least 92.2%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 586-1152 of SEQ ID NO: 7, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-189 of SEQ ID NO: 8 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

63. The polypeptide of embodiment 1 or 62, which comprises amino acids1-189 of SEQ ID NO: 8.

64. An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes a polypeptide having protease activity, and which (a)encodes the polypeptide of any one of embodiments 62-63; (b) hybridizesunder low stringency conditions with (i) nucleotides 586-1152 of SEQ IDNO: 7, (ii) a subsequence of (i) of at least 100 nucleotides, and/or(iii) a complementary strand of (i), or (ii); and/or (c) has a degree ofidentity to nucleotides 586-1152 of SEQ ID NO: 7 of at least 93.6%.

65. The nucleic acid sequence of embodiment 64 which comprisesnucleotides 586-1152 of SEQ ID NO: 7.

66. An isolated nucleic acid sequence produced by (a) hybridizing a DNAunder low stringency conditions with (i) nucleotides 586-1152 of SEQ IDNO: 7; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) acomplementary strand of (i), or (ii); and (b) isolating the nucleic acidsequence.

67. A nucleic acid construct comprising the nucleic acid sequence of anyone of embodiments 64-66 operably linked to one or more controlsequences that direct the production of the polypeptide in a suitableexpression host.

68. A recombinant expression vector comprising the nucleic acidconstruct of embodiment 67.

69. A recombinant host cell comprising the nucleic acid construct ofembodiment 67 or the vector of embodiment 68.

70. A transgenic plant, or plant part, capable of expressing thepolypeptide of any one of embodiments 62-63.

71. A transgenic, non-human animal, or products or elements thereof,being capable of expressing the polypeptide of any one of embodiments62-63

72. A method for producing a polypeptide of any one of embodiments62-63, the method comprising (a) cultivating a recombinant host cell ofembodiment 69 to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide.

73. A method for producing a polypeptide of any one of embodiments62-63, the method comprising (a) cultivating Nocardiopsis sp. DSM 16424;and (b) recovering the polypeptide.

74. Use of at least one protease of any one of embodiments 62-63 (i) inanimal feed; (ii) in animal feed additives; (iii) in the preparation ofa composition for use in animal feed; (iv) for improving the nutritionalvalue of an animal feed; (v) for increasing digestible and/or solubleprotein in animal feed; (vi) for increasing the degree of hydrolysis ofproteins in animal diets; and/or (vii) for the treatment of proteins.

75. A method for improving the nutritional value of an animal feed,wherein at least one protease of any one of embodiments 62-63 is addedto the feed.

76. An animal feed additive comprising (a) at least one protease of anyone of embodiments 62-63; and (b) at least one fat-soluble vitamin,and/or (c) at least one water-soluble vitamin, and/or (d) at least onetrace mineral.

77. The animal feed additive of embodiment 76, which further comprisesamylase; phytase; xylanase; galactanase; alpha-galactosidase; protease,phospholipase; and/or beta-glucanase.

78. An animal feed having a crude protein content of 50 to 800 g/kg andcomprising at least one protease of any one of embodiments 62-63.

79. A method for the treatment of proteins, comprising the step ofadding at least one protease of any one of embodiments 62-63 to at leastone protein or protein source.

80. The method of embodiment 79, wherein soybean is included amongst theat least one protein source.

81. Use of at least one protease of any one of embodiments 62-63 indetergents.

82. An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-189 of SEQ ID NO: 10 ofat least 93.2%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 586-1149 of SEQ ID NO: 9, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-189 of SEQ ID NO: 10 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

83. The polypeptide of embodiment 1 or 82 which comprises amino acids1-189 of SEQ ID NO: 10.

84. An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes a polypeptide having protease activity, and which (a)encodes the polypeptide of any one of embodiments 82-83; (b) hybridizesunder low stringency conditions with (i) nucleotides 586-1149 of SEQ IDNO: 9, (ii) a subsequence of (i) of at least 100 nucleotides, and/or(iii) a complementary strand of (i), or (ii); and/or (c) has a degree ofidentity to nucleotides 586-1149 of SEQ ID NO: 9 of at least 90.3%.

85. The nucleic acid sequence of embodiment 3 or 84 which comprisesnucleotides 586-1149 of SEQ ID NO: 9.

86. An isolated nucleic acid sequence produced by (a) hybridizing a DNAunder low stringency conditions with (i) nucleotides 586-1149 of SEQ IDNO: 9; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) acomplementary strand of (i), or (ii); and (b) isolating the nucleic acidsequence.

87. A nucleic acid construct comprising the nucleic acid sequence of anyone of embodiments 84-86 operably linked to one or more controlsequences that direct the production of the polypeptide in a suitableexpression host.

88. A recombinant expression vector comprising the nucleic acidconstruct of embodiment 87.

89. A recombinant host cell comprising the nucleic acid construct ofembodiment 87 or the vector of embodiment 88.

90. A transgenic plant, or plant part, capable of expressing thepolypeptide of any one of embodiments 82-83.

91. A transgenic, non-human animal, or products or elements thereof,being capable of expressing the polypeptide of any one of embodiments82-83.

92. A method for producing a polypeptide of any one of embodiments82-83, the method comprising (a) cultivating a recombinant host cell ofembodiment 89 to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide.

93. A method for producing a polypeptide of any one of embodiments82-83, the method comprising cultivating Nocardiopsis alkaliphila DSM44657; and recovering the polypeptide.

94. Use of at least one protease of any one of embodiments 82-83 (i) inanimal feed; (ii) in animal feed additives; (iii) in the preparation ofa composition for use in animal feed; (iv) for improving the nutritionalvalue of an animal feed; (v) for increasing digestible and/or solubleprotein in animal feed; (vi) for increasing the degree of hydrolysis ofproteins in animal diets; and/or (vii) for the treatment of proteins.

95. A method for improving the nutritional value of an animal feed,wherein at least one protease of any one of embodiments 82-83 is addedto the feed.

96. An animal feed additive comprising (a) at least one protease of anyone of embodiments 82-83; and (b) at least one fat-soluble vitamin,and/or (c) at least one water-soluble vitamin, and/or (d) at least onetrace mineral.

97. The animal feed additive of embodiment 96, which further comprisesamylase; phytase; xylanase; galactanase; alpha-galactosidase; protease,phospholipase; and/or beta-glucanase.

98. An animal feed having a crude protein content of 50 to 800 g/kg andcomprising at least one protease of any one of embodiments 82-83.

99. A method for the treatment of proteins, comprising the step ofadding at least one protease of any one of embodiments 82-83 to at leastone protein or protein source.

100. The method of embodiment 99, wherein soybean is included amongstthe at least one protein source.

101. Use of at least one protease of any one of embodiments 82-83 indetergents.

102. An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has a degree of identity to amino acids 1-189 of SEQ ID NO: 12 ofat least 83.3%; (b) a polypeptide which is encoded by a nucleic acidsequence which hybridizes under low stringency conditions with (i)nucleotides 586-1152 of SEQ ID NO: 11, (ii) a subsequence of (i) of atleast 100 nucleotides, and/or (iii) a complementary strand of (i), or(ii); (c) a variant of the polypeptide having an amino acid sequence ofamino acids 1-189 of SEQ ID NO: 12 comprising a substitution, deletion,extension, and/or insertion of one or more amino acids; (d) an allelicvariant of (a), or (b); and (e) a fragment of (a), (b), or (d) that hasprotease activity.

103. The polypeptide of embodiment 1 or 102 which comprises amino acids1-189 of SEQ ID NO: 12.

104. An isolated nucleic acid sequence comprising a nucleic acidsequence which encodes a polypeptide having protease activity, and which(a) encodes the polypeptide of any one of embodiments 102-103; (b)hybridizes under low stringency conditions with (i) nucleotides 586-1152of SEQ ID NO: 11, (ii) a subsequence of (i) of at least 100 nucleotides,and/or (iii) a complementary strand of (i), or (ii); and/or (c) has adegree of identity to nucleotides 586-1152 of SEQ ID NO: 11 of at least83.9%.

105. The nucleic acid sequence of embodiment 104 which comprisesnucleotides 586-1152 of SEQ ID NO: 11.

106. An isolated nucleic acid sequence produced by (a) hybridizing a DNAunder low stringency conditions with (i) nucleotides 586-1152 of SEQ IDNO: 11; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii)a complementary strand of (i), or (ii); and (b) isolating the nucleicacid sequence.

107. A nucleic acid construct comprising the nucleic acid sequence ofany one of embodiments 104-106 operably linked to one or more controlsequences that direct the production of the polypeptide in a suitableexpression host.

108. A recombinant expression vector comprising the nucleic acidconstruct of embodiment 107.

109. A recombinant host cell comprising the nucleic acid construct ofembodiment 107 or the vector of embodiment 108.

110. A transgenic plant, or plant part, capable of expressing thepolypeptide of any one of embodiments 102-103.

111. A transgenic, non-human animal, or products or elements thereof,being capable of expressing the polypeptide of any one of embodiments102-103.

112. A method for producing a polypeptide of any one of embodiments102-103, the method comprising (a) cultivating a recombinant host cellof embodiment 109 to produce a supernatant comprising the polypeptide;and (b) recovering the polypeptide.

113. A method for producing a polypeptide of any one of embodiments102-103, the method comprising cultivating Nocardiopsis lucentensis DSM44048, and recovering the polypeptide.

114. Use of at least one protease of any one of embodiments 102-103 (i)in animal feed; (ii) in animal feed additives; (iii) in the preparationof a composition for use in animal feed; (iv) for improving thenutritional value of an animal feed; (v) for increasing digestibleand/or soluble protein in animal feed; (vi) for increasing the degree ofhydrolysis of proteins in animal diets; and/or (vii) for the treatmentof proteins.

115. A method for improving the nutritional value of an animal feed,wherein at least one protease of any one of embodiments 102-103 is addedto the feed.

116. An animal feed additive comprising (a) at least one protease of anyone of embodiments 102-103; and (b) at least one fat-soluble vitamin,and/or (c) at least one water-soluble vitamin, and/or (d) at least onetrace mineral.

117. The animal feed additive of embodiment 116, which further comprisesamylase; phytase; xylanase; galactanase; alpha-galactosidase; protease,phospholipase; and/or beta-glucanase.

118. An animal feed having a crude protein content of 50 to 800 g/kg andcomprising at least one protease of any one of embodiments 102-103.

119. A method for the treatment of proteins, comprising the step ofadding at least one protease of any one of embodiments 102-103 to atleast one protein or protein source.

120. The method of embodiment 119, wherein soybean is included amongstthe at least one protein source.

121. Use of at least one protease of any one of embodiments 102-103 indetergents.

Deposit of Biological Material

The following biological materials have been deposited under the termsof the Budapest Treaty with the DSMZ (Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124Braunschweig, Germany), and given the following accession numbers:

Accession Deposit Number Date of Deposit Nocardiopsis sp. DSM 16424 May24, 2004 Nocardiopsis prasina DSM 15649 May 30, 2003 Nocardiopsisprasina (previously alba) DSM 14010 Jan. 20, 2001

These strains have been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Strain DSM 15649 was isolated in 2001 from a soil sample from Denmark.

The following strains are publicly available from DSMZ:

Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 Nocardiopsisalkaliphila DSM 44657 Nocardiopsis lucentensis DSM 44048

Nocardiopsis dassonvillei subsp. dassonvillei strain DSM 43235 was alsodeposited at other depositary institutions as follows: ATCC 23219, IMRU1250, NCTC 10489.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES Example 1 Cloning and Expression of Three Proteases (L1a, L1b,and L1c)

Reagents and media LB agar Described in Ausubel, F. M. et al. (eds.)“Current protocols in Molecular Biology” John Wiley and Sons, 1995;LB-PG agar LB agar supplemented with 0.5% Glucose and 0.05M potassiumphosphate, pH 7.0 PS-1 10% sucrose, 4% soybean flour, 1% Na₃PO₄—12H₂O,0.5% CaCO₃, and 0.01% pluronic acid TE 10 mM Tris-HCl, pH 7.4 1 mM EDTA,pH 8.0 TEL 50 mg/ml Lysozym in TE-buffer Thiocyanate 5M guanidiumthiocyanate 100 mM EDTA 0.6% w/v N-laurylsarcosine, sodium salt 60 gthiocyanate, 20 ml 0.5M EDTA, pH 8.0, 20 ml H₂O dissolves at 65° C. Cooldown to room temperature (RT) and add 0.6 g N-laurylsarcosine. Add H₂Oto 100 ml and filter it through a 0.2 μ sterile filter. NH₄Ac 7.5MCH₃COONH₄ TER 1 μg/ml RNAse A in TE-buffer CIA Chloroform/isoamylalcohol 24:1

Fermentation of Nocardiopsis Strains

Each of the strains Nocardiopsis dassonvillei subsp. dassonvillei DSM43235, Nocardiopsis prasina DSM 15649, and Nocardiopsis prasina(previously alba) DSM 14010 were grown for 3 days before harvest, in thefollowing medium at 30° C.:

Trypticase 20 g Yeast extract 5 g Ferrochloride 6 mg Magnesium sulfate15 mg Distilled water ad 1000 ml pH adjusted to 9 by addition of sodiumcarbonate.

Preparation of Genomic DNA

Genomic DNA was isolated according to the following procedure:1. Harvest 1.5 ml culture and re-suspend in 100 μl TEL. Incubate at 37°C. for 30 min.2. Add 500 μl thiocyanate buffer and leave at room temperature for 10min.3. Add 250 μl NH₄Ac and leave at ice for 10 min.

4. Add 500 μl CIA and mix.

5. Transfer to a micro-centrifuge and spin for 10 min. at full speed.6. Transfer supernatant to a new Eppendorf tube and add 0.54 volume coldisopropanol.Mix thoroughly.7. Spin and wash the DNA pellet with 70% EtOH.8. Re-suspend the genomic DNA in 100 μl TER.Construction of Bacillus subtilis expression strains Sav-L1a, Sav-L1band Sav-L1c

The coding region for the pro-mature protease L1a (nucleotides 88-1143of SEQ ID NO: 1) was amplified with the following primers 1424 and 1485on genomic DNA isolated from Nocardiopsis dassonvillei subsp.dassonvillei DSM 43235:

Primer 1485 (SEQ ID NO: 14):5′-gcttttagttcatcgatcgcatcggctgcgaccgtaccggccga gccag-3′Primer 1424 (SEQ ID NO: 15):5′-ggagcggattgaacatgcgattactaaccggtcaccagggacag cc-3′

The coding region for the pro-mature protease L1b (nucleotides 88-1149of SEQ ID NO: 3) was amplified with the following primers 1751 and 1753on genomic DNA isolated from from Nocardiopsis prasina DSM15649:

1751 (SEQ ID NO: 16): 5′-gttcatcgatcgcatcggctgtcaccgcacccaccgagcc-3′1753 (SEQ ID NO: 17):5′-ggagcggattgaacatgcgattagctggtgacgaggctgaggttc-3′

The coding region for the pro-mature protease L1c (nucleotides 88-1149of SEQ ID NO: 5) was amplified with the following primers 1755 and 1756on genomic DNA isolated from Nocardiopsis prasina DSM14010:

1755 (SEQ ID NO: 18): 5′-gttcatcgatcgcatcggctgtgaccgcccccgccgag-3′1756 (SEQ ID NO: 19):5′-ggagcggattgaacatgcgattagctcgtgacgaggctgaggttc-3′

Each of these L1a, L1b, and L1c polynucleotides were fused, by PCR, inframe to a heterologous DNA fragment encoding a Say signal peptide (SEQID NO: 13).

Bacillus subtilis strains designated Sav-L1a, Sav-L1b, and Sav-L1c,respectively, were constructed by incorporating these genes (includingthe signal peptide encoding part) by homologous recombination on theBacillus subtilis MB1053 host cell genome (WO 03/95658). The genes wereexpressed under the control of a triple promoter system (as described inWO 99/43835), consisting of the promoters from Bacillus licheniformisalpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene(amyQ), and the Bacillus thuringiensis cryllIA promoter includingstabilizing sequence. The gene coding for Chloramphenicolacetyltransferase was used as marker (described in, e.g., Diderichsen etal., 1993, A useful cloning vector for Bacillus subtilis. Plasmid30:312).

Chloramphenicol resistant transformants were checked for proteaseactivity on 1% skim milk LB-PG agar plates (supplemented with 6 μg/mlchloramphenicol). Some protease positive colonies were further analyzedby DNA sequencing of the insert to confirm the correct DNA sequence, andone strain for each construct was selected.

Fermentation of Bacillus Host Strains

Each of the transformed Bacillus subtilis host strains were fermented ona rotary shaking table (250 r.p.m.) in 500 ml baffled Erlenmeyer flaskscontaining 100 ml PS-1 medium supplemented with 6 μg/ml chloramphenicol,at 37° C. for 16 hours, and at 26° C. for extra 4 days.

Example 2 Cloning and Expression of Protease L2a

The pro-form of a protease encoding gene (nucleotides 88-1152 of SEQ IDNO: 7) was isolated from Nocardiopsis sp. DSM 16424 by the proceduredescribed in Example 1, except for the use of the following primers:

1718 (SEQ ID NO: 20): 5′-gttcatcgatcgcatcggctgcgcccggccccgtcccccag-3′1720 (SEQ ID NO: 21):  5′-ggagcggattgaacatgcgatcagctggtgcggatgcgaac-3′.

The corresponding protease (SEQ ID NO: 8) was designated L2a.

A Bacillus subtilis host strain designated Sav-L2a was constructed, asalso generally described in Example 1, and a chloramphenicol resistant,protease-positive colony selected and analyzed by DNA sequencing of theinsert.

Example 3 Cloning of Two Additional Proteases

The pro-forms of two additional protease encoding genes (nucleotides88-1149 of SEQ ID NO: 9, and nucleotides 88-1152 of SEQ ID NO: 11,respectively) were isolated from Nocardiopsis alkaliphila DSM 44657, andfrom Nocardiopsis lucentensis DSM 44048, respectively, by the proceduredescribed in Example 1, except for the use of primers 1728 and 1763; and1747 and 1749, respectively:

1728 (SEQ ID NO: 22): 5′-gttcatcgatcgcatcggctgcccccgccccccagtc-3′1763 (SEQ ID NO: 23):5′-ggagcggattgaacatgcgattaggtgcgcagacgcaggcccca-3′;1747 (SEQ ID NO: 24): 5′-gttcatcgatcgcatcggctggaaccgtacccaccccccagg-3′1749 (SEQ ID NO: 25): 5′-ggagcggattgaacatgcgattagctggtgcgcagtcgcac-3′

The corresponding proteases (SEQ ID NO: 10 and 12, respectively) weredesignated L2b, and L2c, respectively.

Bacillus subtilis host strains designated Sav-L2b and Sac-L2c,respectively, are constructed, as also generally described in Example 1,and chloramphenicol resistant, protease-positive colonies are selectedand analyzed by DNA sequencing of the inserts.

Example 4 Purification and Characterization of the L1a Protease ProteaseAssays 1) pNA Assay:

pNA substrate: Suc-AAPF-pNA (Bachem L-1400).Temperature: Room temperature (25° C.)Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mMCABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted to pH-values2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0with HCl or NaOH.

20 μl protease (diluted in 0.01% Triton X-100) is mixed with 100 μlassay buffer. The assay is started by adding 100 μl pNA substrate (50 mgdissolved in 1.0 ml DMSO and further diluted 45× with 0.01% TritonX-100). The increase in OD₄₀₅ is monitored as a measure of the proteaseactivity.

2) Protazyme AK Assay:

Substrate: Protazyme AK tablet (cross-linked and dyed casein; fromMegazyme)Temperature: controlled (assay temperature).Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mMCABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted to pH-values2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 with HClor NaOH.

A Protazyme AK tablet is suspended in 2.0 ml 0.01% Triton X-100 bygentle stirring. 500 μl of this suspension and 500 μl assay buffer aremixed in an Eppendorf tube and placed on ice. 20 μl protease sample(diluted in 0.01% Triton X-100) is added. The assay is initiated bytransferring the Eppendorf tube to an Eppendorf thermomixer, which isset to the assay temperature. The tube is incubated for 15 minutes onthe Eppendorf thermomixer at its highest shaking rate (1400 rpm). Theincubation is stopped by transferring the tube back to the ice bath.Then the tube is centrifuged in an icecold centrifuge for a few minutesand 200 μl supernatant is transferred to a microtiter plate. OD₆₅₀ isread as a measure of protease activity. A buffer blind is included inthe assay (instead of enzyme).

Purification

The transformed Bacillus host expressing the L1a protease described inExample 1 was fermented as also described in Example 1, but at 26° C.for 6 days. The culture broth was centrifuged (20000×g, 20 min) and thesupernatants were carefully decanted from the precipitates. The combinedsupernatants were filtered through a Seitz EKS plate in order to removethe rest of the Bacillus host cells. The EKS filtrate was transferred to50 mM H₃BO₃, 5 mM succinic acid, 1 mM CaCl₂, pH 7 on a G25 sephadexcolumn. Solid ammonium sulfate was added to the enzyme solution from theG25 sephadex column to give a 1.6 M final (NH₄)₂SO₄ concentration in theenzyme solution. The enzyme solution was mixed gently with a magneticstirrer during the (NH₄)₂SO₄ addition and the stirring was continued for30 minutes after the addition to bring the system in equilibrium. Thenthe enzyme solution was applied to a Butyl Toyopearl column equilibratedin 100 mM H₃BO₃, 10 mM succinic acid, 2 mM CaCl₂, 1.6 M (NH₄)₂SO₄, pH 7.After washing the column extensively with the equilibration buffer, theprotease was eluted with a linear (NH₄)₂SO₄ gradient (1.6 to 0 M) in thesame buffer. Protease containing fractions were pooled and transferredto 20 mM HEPES, pH 8 on a G25 sephadex column and applied to a Qsepharose FF column equilibrated in the same buffer. After washing thecolumn extensively with the equilibration buffer, the protease waseluted with a linear NaCl gradient (0 to 0.5 M) in the same buffer.Fractions from the column were analysed for protease activity (using theSuc-AAPF-pNA assay at pH 9) and active fractions were further analysedby SDS-PAGE. Fractions with only one band (as judged by a coomassiestained SDS-PAGE gel) were pooled to provide the purified preparationwhich was used for further characterization.

The L1a protease was characterized as described below, in comparisonwith the other protease derived from Nocardiopsis dassonvillei subsp.dassonvillei DSM 43235, prepared as described in WO 2004/111220 (in whatfollows for short designated “the L2 protease”).

pH-Activity, pH-Stability, and Temperature-Activity

The pNA assay was used for obtaining the pH-activity profile as well asthe pH-stability profile. For the pH-stability profile the protease wasdiluted 10× in the assay buffers and incubated for 2 hours at 37° C.After incubation the protease samples were transferred to the same pH(pH 9), before assay for residual activity, by dilution in the pH 9assay buffer. The Protazyme AK assay was used for obtaining thetemperature-activity profile at pH 9. The results are shown in Tables1-3 below.

TABLE 1 pH-activity profile pH L1a protease L2 protease 2 0.00 0.00 30.00 0.00 4 0.02 0.03 5 0.10 0.11 6 0.25 0.21 7 0.38 0.37 8 0.66 0.71 90.97 0.97 10 1.00 1.00 11 0.99 0.94 12 0.94 —

TABLE 2 pH-stability profile pH L1a protease L2 protease 2.0 0.50 1.002.5 0.81 0.95 3.0 0.93 0.97 3.5 0.94 1.01 4.0 0.97 0.98 5.0 0.96 0.976.0 0.95 0.98 7.0 0.99 0.96 8.0 0.97 0.99 9.0 0.93 0.99 10.0 0.94 0.9611.0 0.94 0.94 12.0 0.92 0.84 9.0 and after 2 1.00 1.00 hours at 5° C.

TABLE 3 Temperature activity profile temperature (° C.) L1a protease L2protease 15 0.11 0.01 25 0.17 0.01 37 0.30 0.03 50 0.58 0.09 60 0.900.19 70 1.00 0.63 80 0.34 1.00 90 — 0.35

Other Characteristics

The L1a protease is an alpha-lytic protease like enzyme (peptidasefamily S1E, old notation S2A) which is found to be inhibited by PhenylMethyl Sulfonyl Fluoride (PMSF), and by the Streptomyces SubtilisinInhibitor (SSI). Its relative molecular weight as determined by SDS-PAGEis M_(r)=22 kDa, and the N-terminal sequence: ADIVGGEAY (SEQ ID NO: 26).

Example 5 Specific Activity of the L1a Protease

The purified protease preparation described in Example 4 was used fordetermination of the specific activity. The purity of the preparationwas above 95% when analysed by SDS-PAGE (determined as described inExample 2A in WO 01/58275). The protease sample was divided in two. Onepart was analyzed for protein content (mg/ml) by amino acid analysis,the other part was analysed for protease activity.

Amino Acid Analysis (AAA)/(mq/ml)

The peptide bonds of the protease sample were subjected to acidhydrolysis, followed by separation and quantification of the releasedamino acids on a Biochrom 20 Plus Amino Acid Analyser, commerciallyavailable from Bie & Berntsen A/S, Sandbaekvej 5-7, DK-2610 Roedovre,Denmark, according to the manufacturer's instructions. For the acidhydrolysis, the protein sample was dried in a vacuum centrifuge,resolved in 18.5% (vol/vol) HCl+0.1% (vol/vol) phenol and incubated for16 hr at 110° C. After incubation, the sample was again dried in thevacuum centrifuge, resolved in loading buffer (0.2 M Na-Citrate, pH 2.2)and loaded onto the Biochrom 20 Plus Amino Acid Analyser.

For the quantification, the hydrolysed sample was loaded onto a columnof the cation-exchange resin UltroPac no. 8, Sodium-form, which iscommercially available from Bie & Berntsen A/S, catalogue no.80-2104-15. Buffers of varying pH (pH 1 to pH 8) and ionic strength werepumped through the column according to the manufacturer's instructionsreferred to above, to separate the various amino acids. The columntemperature was accurately controlled, also according to themanufacturer's instructions (from 53° C. to 92° C. and back to 53° C.)in order to ensure the required separation. The column eluent was mixedwith ninhydrin reagent (Bie & Berntsen, catalogue no. 80-2038-07) andthe mixture passed through the high temperature reaction coil of theAmino Acid Analyser. In the reaction coil, ninhydrin reacted with theamino acids to form coloured compounds, the amount of which was directlyproportional to the quantity of amino acid present.

Protease Activity Assay (AU/ml)

Denatured haemoglobin (0.65% (w/w) in 6.7 mM KH₂PO₄/NaOH buffer, pH7.50) was degraded at 25° C. for 10 minutes by the protease, andundigested haemoglobin was precipitated with trichloroacetic acid (TCA)and removed by filtration. The TCA-soluble haemoglobin degradationproducts in the filtrate were determined with Folin & Ciocalteu's phenolreagent, which gives a blue colour with several amino acids. Theactivity unit (AU) was measured and defined by reference to an ALCALASE™standard. A detailed description of the assay, as well as a sample ofthe ALCALASE™ standard, is available on request from Novozymes A/S,Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark (assay no.EB-SM-0349.02/01).

The specific activity was calculated as: Specific activity(AU/g)=(Activity (AU/ml)/AAA (mg/ml))×1000 (mg/g).

The specific activity of the L1a protease was 49.8 AU/g, as compared tothe specific activity of the protease derived from Nocardiopsis sp. NRRL18262 of 38.3 AU/g.

Example 6 Purification and Characterization of the L2a Protease

The transformed Bacillus host expressing the L2a protease described inExample 2 was fermented as described in Example 1, but at 30° C. for 5days. The culture broth was centrifuged (20000×g, 20 min) and thesupernatants were carefully decanted from the precipitates. The combinedsupernatants were filtered through a Seitz EKS plate in order to removethe rest of the Bacillus host cells. The EKS filtrate was transferred to50 mM H₃BO₃, 5 mM succinic acid, 1 mM CaCl₂, pH 7 on a G25 sephadexcolumn and applied to a bacitracin silica column equilibrated in thesame buffer. After washing the bacitracin column extensively with theequilibration buffer, the protease was step-eluted with 100 mM H₃BO₃, 10mM succinic acid, 2 mM CaCl₂, 1 M NaCl, 25% isopropanol, pH 7. Thebacitracin eluate was transferred to 50 mM H₃BO₃, 10 mM CH₃COOH, 1 mMCaCl₂, pH 4.5 on a G25 sephadex column and applied to a S sepharose HPcolumn equilibrated in the same buffer. After washing the columnextensively with the equilibration buffer, the protease was eluted witha linear NaCl gradient (0 to 0.5 M) in the same buffer. Fractions fromthe column were analysed for protease activity (using the Protazyme AKassay at 37° C. and pH 9) and active fractions were further analysed bySDS-PAGE. Fractions with only one band was (as judged by a coomassiestained SDS-PAGE gel), were pooled to provide the purified preparationwhich was used for further characterization.

The L2a protease was characterized as described in Example 4 above, incomparison with a known protease derived from Nocardiopsis sp. NRRL18262 (for short designated “Protease 10”). The results are shown inTables 4-6 below.

TABLE 4 pH-activity profile pH L2a protease Protease 10 2 0.00 — 3 0.000.00 4 0.02 0.02 5 0.10 0.07 6 0.22 0.21 7 0.41 0.44 8 0.75 0.67 9 0.970.88 10 0.99 1.00 11 1.00 0.93 12 0.85 —

TABLE 5 pH-stability profile pH L2a protease Protease 10 2.0 0.67 0.782.5 0.93 1.00 3.0 0.95 1.03 3.5 0.96 0.98 4.0 0.97 0.99 5.0 0.94 1.026.0 0.95 1.00 7.0 0.97 1.01 8.0 0.96 0.98 9.0 0.95 0.99 10.0 0.96 0.9911.0 0.90 0.86 12.0 0.60 — 9.0 and after 2 1.00 1.00 hours at 5° C.

TABLE 6 Temperature activity profile Temperature (° C.) L2a proteaseProtease 10 15 0.02 0.02 25 0.02 0.02 37 0.05 0.07 50 0.13 0.20 60 0.310.51 70 0.79 1.00 80 1.00 0.39 90 0.28 —

Other Characteristics

The L2a protease is an alpha-lytic protease like enzyme (peptidasefamily S1E, old notation S2A) which is found to be inhibited by PhenylMethyl Sulfonyl Fluoride (PMSF). Its relative molecular weight asdetermined by SDS-PAGE is M_(r)=20 kDa, and the N-terminal sequence:ANIIGGLAYT (SEQ ID NO: 27).

Example 7 Melting Temperature of the L2a Protease Differential ScanningCalorimetry (DSC)

DSC was used to determine temperature stability at pH 7.0 of the L2aprotease derived from Nocardiopsis sp. DSM 16424. The protease waspurified as described in Example 6 and dialysed over night at 4° C.against 10 mM sodium phosphate, 50 mM sodium chloride, pH 7.0 and run ona VP-DSC instrument (Micro Cal) with a constant scan rate of 1.5° C./minfrom 20 to 100° C. Data-handling was performed using the MicroCal Originsoftware.

The resulting denaturation or melting temperature (T_(m) or T_(d)), was78.2° C. The T_(m) for Protease 10 is 76.5° C.

Example 8 Performance of the L2a Protease in a Monogastric In VitroDigestion Model

The performance of the purified L2a protease described in Example 6 wastested in an in vitro model simulating the digestion in monogastricanimals, in comparison with the known protease derived from Nocardiopsissp. NRRL 18262 (“Protease 10”). In particular, the protease was testedfor its ability to improve solubilization and digestion of maize/-SBM(maize/-soybean meal) proteins. The in vitro system consisted of 18flasks in which maize/-SBM substrate was initially incubated withHCl/pepsin—simulating gastric digestion—and subsequently withpancreatin—simulating intestinal digestion. Eight of the flasks weredosed with the protease at the start of the gastric phase whereas theremaining ten flasks served as blanks. At the end of the intestinalincubation phase samples of in vitro digesta were removed and analyzedfor solubilized and digested protein.

Outline of In Vitro Digestion Procedure

Simulated Time digestion Components added pH Temperature course phase 10g maize/-SBM substrate 3.0 40° C. t = 0 min Mixing (6:4), 41 ml HCl(0.105M) 5 ml HCl (0.105M)/pepsin 3.0 40° C t = 30 min Gastric (3000 U/gsubstrate), 1 ml digestion protease (to provide 100 mg protease enzymeprotein per kg of substrate) 16 ml H₂O 3.0 40° C. t = 1.0 hour Gastricdigestion 7 ml NaOH (0.39M) 6.8 40° C t = 1.5 hours Intestinal digestion5 ml NaHCO₃ (1M)/ 6.8 40° C. t = 2.0 hours Intestinal pancreatin (8 mg/gdiet) digestion Terminate incubation 7.0 40° C. t = 6.0 hours

Conditions

Substrate: 4 g SBM, 6 g maize (premixed)pH: 3.0 stomach step/6.8-7.0 intestinal stepHCl: 0.105 M for 1.5 hours (i.e., 30 min HCl-substrate premixing)pepsin: 3000 U/g diet for 1 hourpancreatin: 8 mg/g diet for 4 hourstemperature: 40° C.

Replicates: n Solutions 0.39 M NaOH 0.105 M HCl

0.105 M HCl containing 6000 U pepsin per 5 ml1 M NaHCO₃ containing 16 mg pancreatin per ml

125 mM NaAc-buffer, pH 6.0 Enzyme Protein Determinations

The amount of protease enzyme protein (EP) is calculated on the basis ofthe A₂₈₀ values and the amino acid sequences (amino acid compositions)using the principles outlined in Gill & von Hippel, 1989, AnalyticalBiochemistry 182: 319-326.

Experimental Procedure for In Vitro Model

The experimental procedure was according to the above outline. pH wasmeasured at time 1, 2.5, and 5.5 hours. Incubations were terminatedafter 6 hours and samples of 30 ml were removed and placed on ice beforecentrifugation (10000×g, 10 min, 4° C.). Supernatants were removed andstored at −20° C.

Analysis

All samples were analyzed for content of solubilized and digestedprotein using gel filtration.

Estimation of Solubilized and Digested Protein

The content of solubilized protein in supernatants from in vitrodigested samples was estimated by quantifying crude protein (CP) usinggel filtration HPLC. Supernatants were thawed, filtered through 0.45 μmpolycarbonate filters and diluted (1:50, v/v) with H₂O. Diluted sampleswere chromatographed by HPLC using a Superdex Peptide PE (7.5×300 mm)gel filtration column (Global). The eluent used for isocratic elutionwas 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl. Thetotal volume of eluent per run was 26 ml and the flow rate was 0.4ml/min. Elution profiles were recorded at 214 nm and the total areaunder the profiles was determined by integration. To estimate proteincontent from integrated areas, a calibration curve (R²=0.9993) was madefrom a dilution series of an in vitro digested reference maize/-SBMsample with known total protein content. The protein determination inthis reference sample was carried out using the Kjeldahl method(determination of % nitrogen; A.O.A.C. (1984) Official Methods ofAnalysis 14th ed., Washington D.C.).

The content of digested protein was estimated by integrating thechromatogram area corresponding to peptides and amino acids having amolecular mass of 1500 Dalton or below (Savoie et al., 1986, DialysisCell For The In-vitro Measurement Of Protein Digestibility. J. Food Sci.51, 494-498; Babinszky et al., 1990, An In-vitro Method for Predictionof The Digestible Crude Protein Content in Pig Feeds. J. Sci. Food Agr.50: 173-178; Boisen et al., 1991, Critical Evaluation of In-vitroMethods for Estimating Digestibility in Simple-Stomach Animals.Nutrition Research Reviews 4: 141-162). To determine the 1500 Daltondividing line, the gel filtration column was calibrated using cytochromeC (Boehringer, Germany), aprotinin, gastrin I, and substance P (SigmaAldrich, USA), as molecular mass standards.

Results

The results shown in Table 7 below indicate that the L2a protease, likeProtease 10, significantly increased the level of soluble and digestibleprotein relative to the blank. Furthermore, the L2a protease appears toat least numerically improve the level of digestible protein as comparedto the known Protease 10.

TABLE 7 Solubilized and digested crude protein Relative to blank EnzymeN % digestible CP CV % % soluble CP CV % Blank 10 100.0 ^(a) 5.5 100.0^(a) 4.4 L2a protease 3 116.1 ^(b) 0.7 107.2 ^(b) 1.1 Protease 10 5112.1 ^(b) 1.0 110.2 ^(b) 0.6 Different letters within the same columnindicate significant differences (1-way ANOVA, Tukey-Kramer test, P <0.05). SD = Standard Deviation. % CV = Coefficient of Variance =(SD/mean value) × 100%

Example 9 Animal Feed and Animal Feed Additives

An animal feed additive comprising protease L2a of the invention, in theform of a vitamins and mineral premix, is composed as shown in Table 8below. The vitamins and the carotenoids are commercially available fromDSM Nutritional Products. All amounts are in g/kg.

TABLE 8 Premix composition Vitamin A ROVIMIX A 500 4.00 Vitamin D3ROVIMIX D3 500 1.00 Vitamin E ROVIMIX E 50 Ads 8.00 Vitamin B2 ROVIMIXB2 80-SD 1.0 CAROPHYLL Yellow 10.0 Choline chloride 50%, min. 300.0Minerals Mn Oxide 60.0 Zn Oxide 12.0 Fe Sulphate monohydrate 20.0 CuOxide 2.0 Co Sulphate 0.2 Enzyme Protease L2a (enzyme protein) 10.0Wheat middlings 571.8

The Premix of Table 8 is included in a diet for layers with acomposition as shown in Table 9 below. The amount of each ingredient isindicated in % (w/w). The concentration in the diet of the L2a proteaseis 100 mg protease enzyme protein per kg of the diet.

TABLE 9 Diet for layers Maize 55.00 Wheat 10.00 Oat 7.50 Soya 20.00Limestone 7.50 Premix of Table 8 1.00

1-20. (canceled)
 21. An isolated nucleic acid sequence comprising anucleic acid sequence which encodes a polypeptide having proteaseactivity, wherein the polypeptide is selected from the group consistingof: (a) a polypeptide having a sequence identity of at least 90% to thesequence of amino acids 1-192 of SEQ ID NO: 2, or which is a fragment ofthe sequence of amino acids 1-192 of SEQ ID NO: 2, that has proteaseactivity; (b) a polypeptide having a sequence identity of at least 90%to the sequence of amino acids 1-192 of SEQ ID NO: 8, or which is afragment of the sequence of amino acids 1-192 of SEQ ID NO: 8, that hasprotease activity; (c) a polypepdite having a sequence identity of atleast 90% to the sequence of amino acids 1-189 of SEQ ID NO: 10, orwhich is a fragment of the sequence of amino acids 1-189 of SEQ ID NO:10, that has protease activity; and (d) a polypeptide having a sequenceidentity of at least 90% to the sequence of amino acids 1-189 of SEQ IDNO: 12, or which is a fragment of the sequence of amino acids 1-189 ofSEQ ID NO: 12, that has protease activity.
 22. A nucleic acid constructcomprising the nucleic acid sequence of claim 21, wherein the nucleicacid sequence is operably linked to one or more control sequences thatdirect the production of the polypeptide in a recombinant host cell. 23.A recombinant expression vector comprising the nucleic acid construct ofclaim
 22. 24. A recombinant host cell comprising the nucleic acidconstruct of claim
 22. 25. A method for producing a polypeptide havingprotease activity, comprising (a) cultivating a recombinant host cell ofclaim 24 under conditions for producing the polypeptide; and (b)recovering the polypeptide.